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LANCE Protein-Protein Interaction Assays

 

Overview


The LANCE® TR-FRET technology is commonly used to study protein-protein and protein-peptide interactions. The most common biomolecular interaction assay format uses a biotinylated peptide, a tagged (GST, c-myc, 6xHis) protein, a Eu-anti-tag antibody, and a ULight™-streptavidin or APC (allophycocyanin)-streptavidin. This format is often used to study nuclear receptors, using recombinantly-produced soluble regions of the receptor with a biotinylated peptide or small molecule. LANCE can also be used to study DNA-protein interactions, small molecule-protein interactions, etc.

Because the energy transfer distance in any FRET assay is somewhat limited (~10 nm), we recommend using our Alpha technology if you would like to study larger protein-protein interactions (spanning distances up to 200 nm or more).

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What do I need to run this assay?


Required reagents available from PerkinElmer:

  • LANCE Europium-labeled assay component (see products and catalog numbers list below; PerkinElmer also offers custom labeling services, or you can Label your own LANCE reagent)
  • Acceptor fluorophore-labeled assay component (see products and catalog numbers list below; PerkinElmer also offers custom labeling services)
  • Microplate (We recommend white OptiPlate™ or ProxiPlate™ microplates.)
  • TopSeal™-A adhesive plate seal

  • If you will be using BSA in your assay buffer, we recommend 7.5% BSA solution (PerkinElmer # CR84-100)


Required reagents available from various suppliers:


Instrumentation/Equipment:

  • A TRF-capable plate reader (We recommend the PerkinElmer EnVision® or VICTOR® Plate Reader.)

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Products and catalog numbers


View a listing of relevant catalog numbers.

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Assay design


General recommendations

  • Always use the donor Europium chelate to label a binding reagent that has good purity and affinity
  • Do not label impure reagents, such as polyclonal antibodies, with the Europium donor fluorophore - impure reagents should be labeled with the acceptor fluorophore instead
  • If you are measuring a low affinity binding interaction, use the acceptor reagent in excess to push the binding equilibrium. You can use higher concentrations of acceptor fluorophore (as opposed to donor fluorophore). Increasing the donor fluorophore concentration would increase your assay background.
  • Try to get good proximity between the donor and acceptor fluorophores; TR-FRET efficiency is very much distance-dependent
  • Be aware of buffer requirements related to some chelates.
  • Use appropriate high sensitivity plate readers with optimized filters and windows to measure the assay. We recommend using a VICTOR or Envision Plate Reader, or a ViewLux Reader for ultra high throughput.

 

Donor fluorophore concentration

Although Europium chelates are perfect donors for energy transfer, there still remains a tiny signal cross-talk interference from donor to the used acceptor signal window. This is typical for all Eu chelates/cryptates, regardless of the supplier. For example, the W1024-Eu chelate gives about 0.3% interference to the 665 nm window. With the W8044-Eu chelate, cross-talk is about 0.6%. As a general rule, the binding percentage for the Europium reagent needs to be 1% or more to get feasible S/N. We typically recommend using 0.1-10 nM of the Europium fluorophore-labeled reagent. Higher concentrations will result in increased background.

Table 1 (further below) gives examples of concentrations used in different assays.

 

Acceptor fluorophore concentration

As the acceptor fluorophore will not give any direct TR-FRET signal on its own, there is a larger freedom in concentration of the acceptor-labeled reagent, which generally can be higher (up to about 100 nM). Acceptor label and labeled components give unspecific interference only by diffusion limit. The diffusion related background is thus molecular weight dependant, and high molecular weight reagents can be used at higher molar concentrations than respective small molecular weight reagents. For example, APC-SA (streptavidin labeled with allophycocyanin), can be used at rather high molar concentration.

 

Proximity

FRET generally requires relatively close proximity to produce good signal. R0 values are generally in the range of 60 – 100 Å (6 – 10 nm). R0 is defined as a distance giving 50% ET (energy tranfer) efficiency. Lower ET efficiency is also useful in assays, but the signal is inversely related to the sixth power of distance. Accordingly, when donor fluorophore-acceptor fluorophore distance increases to 2xR0 value, ET efficiency drops to 0.8%. Due to their long excited state, lanthanides have specific advantages (e.g. in respect to orientation factor). Also, conformational tumbling (e.g. with flexible peptides) allows very efficient ET efficiency regardless of theoretical distance (i.e., ET takes place at any moment the distance is minimal during the excited state).

Due to the proximity requirement, direct assays would technically be recommended (i.e. assays where the binding partners are directly labeled with Europium donor chelate and small molecular ULight acceptor fluorophore). Due to technical difficulties in direct labeling of specific reagents, most of the applications (see Table 1) rely on secondary reagents, such as streptavidin and suitable anti-tag antibodies. In an example study of herpes virus entry mediator (receptor-ligand), direct labeling gave a higher S/N (4.4) compared to indirect labeling using Eu-protein A and APC-streptavidin (2.6). However, the indirect method was chosen for screening (Moore 1999). We do offer custom labeling services if you would like for us to directly-label your proteins or peptides for you - contact information is at the bottom of this page.

Due to the short decay time of acceptor fluorophores, one single acceptor fluorophore dye molecule can theoretically function as an acceptor for a number of donor fluorophore dye molecules. In practice, however, the major response will come from the donor fluorophore-acceptor fluorophore pair having the shortest proximity. Therefore, the reagents are generally labeled with a suitable number of labels, depending mainly on their tolerance for substitutions. Antibodies (which are larger compared to other types of biomolecules), for example, are generally labeled with about 5 – 8 Eu per IgG.

 

Buffers

0.1% BSA is typically added to the binding buffer. We recommend that you use our DTPA-purified BSA solution as a source for BSA (Cat. No. CR84-100). You may need to optimize your buffer composition to achieve best results. Various Europium chelates have various tolerances for certain buffer components. For example the W1024-Eu chelate is optimized for TR-FRET and labeling, but should be used in an assay buffer at neutral or basic pH, can withstand EDTA for only limited time, and is quenched by heavy metallic cations (such as Mn2+). Our W8044-Eu chelate is more stable and can stand rather high concentrations of EDTA. Both chelates have negative net charge, and if the assay requires the use of Mn2+, it is necessary to complex it with excess of EDTA in the detection mixture. If you are working with the W1024 chelate, we recommend that you avoid using phosphate buffers in your assay. Phosphates can decrease the signal for the W1024 Europium chelate. View more information on Europium chelate structure and tolerance.

 

Controls

As both reagents (donor fluorophore and acceptor fluorophore) potentially affect both specific signal and background, control experiments should have the same labels (either avoiding one specific binding partner, or using excess of unlabeled competing component).

 

Applications

Table 1 below gives some examples of LANCE protein-protein applications, identifying the reagents and their concentrations in the assay. The majority of applications relate to nuclear receptors (coactivator recruitment) and receptor activation (dimerization). The citations section provides further applications in different fields of biodiscovery.


Table 1. Applications of LANCE protein-protein interaction assays.

AssayEuropium donor fluorophore reagentConcentration of donor reagentAcceptor fluorophore reagentConcentration of acceptor reagentReference
Direct labeling     
HVEM – HVEM-REu-HVEM APC-Ab Moore 1999
FXR - SRCEu-GST-FXR-LBD8 nMAPC-SRC16 nMUrizar 2002
IL-13 – IL-13-REu-IL1320 nMAccept-IL-R0-1000Yang 2008
     
Nuclear receptors     
Estrogen receptorbio-ERE + EU-SA10 nM; 2.5 nMAcceptor-SRC10 nMLikhite 2006
Estrogen receptorER-LBD-FLAG + Eu-anti-FLAG5 nM; 1 nMbio-coact (src) + APC-SA100 nM; 30 nMLiu 2003
Miner.corticoid-RGST-MR-LBD + Eu-anti-GST2 nMbio-peptide + APC-SA100 nM; 60 nMHultman 2005
PPARHIS-PPAR-LBD + Eu-anti-HIS20 nM; 1 nMbio-peptide + APC-SA500 nM 100 nMPochetti 2007
PPAREu-LxxLL bio-PPAR-LBD + APC-SA Shearer 2008
PXRHIS-PXR + Eu-anti-HIS20 nMbio-coactivator + APC-SA300 nMMitro 2007
SREBPHis-SREBP + Eu-anti-HIS1 nMGST-SREBP + APC-anti-GST30 nMNajima 2005
      
Dimer / Oligomer     
Histidine receptorHR-c-myc + Eu-anti-c-myc5 nMbio-HR + APC-SA15 nMBakker 2004
CXCR1 /4Eu-anti-c-myc5 nMAPC-anti-FLAG15 nMWilson 2005
Dopamine receptorEu-anti-FLAG2.5 nMAPC-anti-FLAG2.5 nMGazi 2003
      
Protein-Protein, Receptor-Ligand     
Cks1 – SKp2Eu-anti-FLAG0.32 μg/mlAPC-anti-GST0.2-0.4 μg/mlHuang 2005
IL13 – IL13-BPIL-13-c-myc + Eu-anti-c-myc50 nM; 5 nMbio-IL13 R + APC-SA100 nM; 20 nMYang 2006
HVEMHVEM-Fc + Eu-ProtA10 nM; 10 nMbio-HVEM-L + APC-SA50 nM; 60 nMMoore 1999

*When two concentrations are listed instead of one, the first value refers to the concentration of the tagged protein, and the second value refers to the concentration of the fluorophore-labeled reagent.

 

Assay optimization


  1. Cross-titration of donor and acceptor fluorophore
  2. Cross-titration of two biomolecules of interest
  3. Biotin:streptavidin ratio optimization, if applicable

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Guides


Quick Start Guide for LANCE Protein-Protein Interactions

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Citations


View a brief list of LANCE biomolecular interaction citations.

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Tips


  • In TR-FRET assays the donor fluorophore-labeled reagent is typically used at a lower concentration than the acceptor fluorophore-labeled reagent to minimize background. For example, in the assay developed by Wu et al. (full citation below), GST-FXR (0.5 nM), biotinylated SRC-1 peptide (5 nM), streptavidin conjugated APC (2 nM), and Europium-labeled anti-GST antibody (1 nM) are used. Wu, X., Sills, M.A. & Zhang, J. Further Comparison of Primary Hit Identification by Different Assay Technologies and Effects of Assay Measurement Variability. J Biomol Screen 10, 581-589 (2005). Link
  • Keep in mind the binding affinities for the association of your peptide or protein with the fluorophore-labeled reagent. The streptavidin-biotin interaction is a fairly strong interaction. You might see a hook effect if the ratio of biotin to streptavidin exceeds 4:1.
  • If using streptavidin, we recommend starting with a biotin:strepatavidin ratio of 4:1. Streptavidin is a tetramer. This ratio can later be optimized at the final stages of assay development.
  • Weaker interactions may require the addition of more of one binding partner to push the equilibrium to more protein-protein complex. You can leverage the streptavidin valency for biotin to keep your background relatively low while increasing the amount of biotinylated peptide.

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Custom labeling and custom assay development services at PerkinElmer


PerkinElmer offers custom labeling services as well as custom assay development. If you are interested in having your biomolecule custom-labeled, or in custom assay development, please contact our custom teams:

ON>POINT® Custom Labeling and Conjugation Services 

ON>POINT® Custom Assay Development Services

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